"Co-Administration of Ketamine and Ethanol Induced Tau Hyperphosphorylation and Amyloid Accumulation in the Hippocampus and Striatum of Rats"

Methods: Here, we investigated the effects of different length exposure of ketamine with and without ethanol on mRNA expressions of the tau hyperphosphorylation and β-amyloid (Aβ) deposition related genes in the rat’s hippocampus and striatum. We also examined the effects of the combined drug treatment on the phosphorylating tau protein at multiple sites and Aβ accumulation, as well as related protein levels in the same brain areas of the rats.


Introduction
Derived from phencyclidine (piperidine, PCP), ketamine acts as a noncompetitive blocker of N-methyl-D-aspartate (NMDA). The psychological effects of ketamine have been defined as relating to sensory deprivation, mood elevation, and cognitive impairments [1]. Ketamine has been applied in medical use and psychological research for alleviating adverse symptoms stemming from the pathophysiology of depression. On the other hand, it has become a street drug used recreationally in many countries, raising concerns of public health officials worldwide [2]. The prevalence of ketamine use was found to be 1.5% among 12th-grade students in the US; in mainland China, the use of synthetic drugs has increased from 5.6% to 53.8% (2003 to 2010) [3]. Upon three consecutive days of the administration, recreational ketamine users display semantic memory impairment and dissociative and schizotypal symptomatology, associated with ketamine's action through NMDA receptors [4,5]. Studies have also shown that ketamine's use at an anesthetic or sub-anesthetic dose could impair the cognitive processes involved in learning and memory, suggesting that nonassociative taste memories may be disrupted by NMDA receptor blockade [6,7]. Current animal research indicates that repeated ketamine exposure augmented NMDA receptor subunit gene expression, notably subunit 1 (NMDAR1 or GluN1). Full sequencing of NMDA receptor genes may help to understand the individual's vulnerability to ketamine abuse [8].
NMDA receptors, which are vital for learning and memory, are known to be involved in neurotoxic events. NMDA receptors are also hypothesized to play instrumental roles in the pathophysiology of Alzheimer's disease (AD) [9]. In the prefrontal and entorhinal cortical sections of mouse and monkey brains, significant increases in hyperphosphorylated tau protein were observed after 6 months of ketamine administration. [10] Several kinases and phosphatases have been reported as contributing to tau hyperphosphorylation, including the kinases glycogen synthase kinase-3 (GSK-3) and cyclin-dependent kinase 5 (CDK-5). In contrast, protein phosphatase 2A (PP2A) appears to be the most plausible phosphatase involved in the abnormal posttranslational modification -phosphorylation and methylation [11,12]. Besides, ketamine reduced β-amyloid protein (Aβ) degradation via suppressing neprilysin expression through the dephosphorylating of p38 mitogen-activated protein kinase (MAPK) mediated pathway, which is thought to promote Aβ deposition in patients with AD [13]. There are two pathophysiological hallmarks of AD: the deposition of intracellular neurofibrillary tangles that are aggregates of hyperphosphorylated tau and extracellular plaques that are deposits of amyloid-beta (Aβ) [14]. The question is whether the cognitive impairment of ketamine abuse is related to similar biological events relevant to AD pathogenesis.
Our group recently found a significant increase in dopamine (DA) concentration of rats' striatum accounted for drug-paired place preference induced by ketamine and ethanol co-administration [15]. Aβ stimulates dopamine release from dopaminergic axons in the anterior cingulate cortex, and excessive dopamine over activates D1 receptors in fast-spiking interneurons, thus contributing to gamma-aminobutyric acid (GABA) inhibitory and excitation/ inhibition imbalance caused by Aβ [16]. These exciting results bring up several points of investigation: (a) Whether the aggregation of hyperphosphorylated tau in the rat brains is mediated through the GSK-3 pathway; [17] (b) If the deposition of Aβ in the specific brain area of rats is associated with the activity of β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1); [18] (c) The degree to which the NMDAR subunit expression is modulated in the rat central nervous system after recreational ketamine abuse.
As ketamine and ethanol are recreationally consumed together, we focused on the synergic effects of co-administration of ketamine with ethanol on the rat striatum and hippocampus. In this study, we measured the expression profile of the tau hyperphosphorylation and Aβ deposition-related genes (GSK-3β, Protein phosphatase 2 (PP2A) and BACE1), the levels of tau phosphorylation at serine residues 396/ 404 (Ser 396/404) and threonine residues 231 (Thr 231), and the phosphorylation of GSK-3β. We also examined the expression of NMDAR subunits (NMDAR1 or GluN1, NMDAR2B or GluN2B), Aβ expression, and total tau protein. To our knowledge, this study is the first to scrutinize the cognitive impairment induced by the co-administration of ketamine and ethanol from an angle of AD-related biological events. The two control groups (Control and Ethanol) received the same dose of ethanol and saline. The experimental conditions of each group can be seen in (Table 1). The body weights of the rats were measured weekly, and doses were adjusted appropriately [12]. rats

Quantitative Real-Time Polymerase Chain Reaction
qRT-PCR was performed as previously reported [15]. Each  analysis. Differential gene expressions between the drug and saline groups were calculated using the 2 -△△CT method with GAPDH as an endogenous control. Relative quantification of GSK-3β, PP2A, and BACE1 mRNA expression was achieved as previously described [21].

Statistical Analyses
All data are expressed as the means ± SEM. All statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software Inc., USA) for Windows. Subsequent analyses involved repeated measures analysis of variance (rmANOVA) in confirming the interactions between the length of exposure and different treatments. When significant, analyses of variance were followed by group comparisons using Tukey's post-hoc-tests. p < 0.05 was considered statistically significant.

Results
As recapitulated in (Supplementary Table 1  BACE1 expression was not altered, except for a decrease in the K30 group by 14.90% without statistical significance ( Figure 1C).
Significant time-dependent decrease of GSK3β, PP2A, and BACE1 mRNA expression was only found for the K group. In the striatum of 3-month groups, as shown in (Figure 2), compared with the control, we found an increase in GSK3β mRNA expression in the K30 group ( (Figure 2A), 53.04%, p = 0.0007 versus K30; 6 months).
Additionally, a critical increase in BACE1 mRNA expression was noticed in the K30+E group (( Figure 2C) On the contrary, we observed the opposite alteration of p-GSK-3β protein levels in each drug administration group corresponding to the variation of GSK-3β protein but without statistical significance compared with controls ( Figure 3E). There was no visible alteration of PP2A protein expression observed in all drug treatment groups ( Figure 3F). Western blot analysis showed that similar changes of p-Tau (Thr231) levels in the striatum across different groups were observed after 3 and 6 months of drug administration.
However, the results did not reach statistical significance ( Figure   4A). Surprisingly, compared with controls, the highest level of tau phosphorylation at serine 396 was observed in the E group after 3 months of drug administration (increased by 153.4%, p = 0.0001).
As well, ketamine with or without ethanol induced the elevated A. Quantitative analysis of p-Tau (Thr231) protein.
D. The levels of GSK-3β was measured.
E. The levels of p-GSK-3β was measured.
F. The levels of PP2A was measured.
The compensatory alterations of p-GSK-3β to GSK-3β were found in the striatum ( Figure 4E). The expressions of p-GSK-3β were increased after 3 months of drug administration (K group: A. Quantitative analysis of p-Tau (Thr231) protein.
D. The levels of GSK-3β was measured.
E. The levels of p-GSK-3β was measured.
F. The levels of PP2A was measured.
G. The levels of proteins was detected by Western blot. The data was expressed as mean ± SEM. (*p < 0.05, **p < 0.01 versus controls and #p < 0.05, ##p < 0.01 versus drugs administration group).  We observed the intense brown positive staining (showed with the black arrow in (Figure 7) that indicated the similar augment of Aβ and total tau in the striatum when exposure to ketamine alone and ketamine combined with ethanol for 3 months (p < 0.0001 versus controls, ( Figure 8A) and ( Figure 8B). In contrast to the changes in the hippocampus, ketamine with or without ethanol treatment at the same time promoted appreciable increases in and NMDAR1 protein levels (p < 0.0001, ( Figure 8C). In reverse, 3 months of treatment with Ketamine and Ketamine plus ethanol were found to be effective in reducing expression of NMDAR2B (p < 0.0001 versus controls, ( Figure 8D). 6 months of treatment with ketamine substantially increased expression of Aβ, total tau, and NMDAR1 (p < 0.0001 versus controls, (Figures 8A-8C). In contrast to an increase of NMDAR2B protein level in the hippocampus of the K30+E group, we observed an exceptional decrease of NMDAR2B expression in the striatum of the K30+E group (p < 0.001 versus K30 group, ( Figure 8D). Additionally, we noticed that time-dependent decreased expressions of Aβ, total tau, and NMDAR1 were found for K and K+E groups, but time-dependent increased expressions of NMDAR2B were found for K and K+E groups.  A. Semi-quantitative analysis of Aβ protein.
B. Semi-quantitative analysis of total tau protein.
C. Semi-quantitative analysis of NMDAR1.

Discussion
To better comprehend chronic ketamine users' cognitive impairment, we considered the influence of other drugs. We explored possible synergistic interactions between ketamine and other illicit drugs on the accumulation Aβ as well as hyperphosphorylation of tau. We established rat models that treated them with ketamine only and ketamine combined with ethanol to mimic recreational ketamine abuse. Drug abusers self-administer ketamine by inhaling (60-250mg), consuming orally (200-300mg), injecting intramuscularly (75-125 mg), or injecting intravenously (50-100 mg) [23]. In this study, rat models have been treated with ketamine at the sub-anesthetic dose of 30 mg/kg. We chose 3 and 6 months as drug administration lengths according to suggested long-term drug treatment [24,25].

Effect of Different Ketamine Treatments on Tau Hyperphosphorylation and Total Tau
Hyperphosphorylated tau is the major component of the cells and in the hippocampus of SD rats, which is mediated by abnormal CDK5-regulated tau phosphorylation [27]. Additionally, tau phosphorylated at threonine 231 differentiated between AD and frontotemporal dementia. 11 Interestingly, we found a significant increase in tau phosphorylated expression at serine 404 after treating rats with Ketamine 30 mg/kg for short and long-term, which was probably mediated mutually by GSK-3β and CDK5 but needed to be further clarified. To our knowledge, this was the first report on ketamine-induced alteration of serine 404 sites of tau phosphorylation in the striatum. As the activity of GSK-3β is prevented via the phosphorylation of GSK-3β at Ser 9, [28] the reverse alterations of p-GSK-3β (Ser9), compared with GSK-3β, were observed in the hippocampus and striatum of all current rat models. PP2A balances tau phosphorylation directly via dephosphorylation of tau and indirectly via the upregulation of GSK-3β in the brain [29]. In the current study, we found a significant increase of phosphorylated tau protein at Ser 396 accompanied by upregulation of GSK-3β and PP2A expression and inhibited the activity of p-GSK-3β (Ser 9) in the hippocampus of the rats treated with ketamine and ethanol for 6 months.
The alterations probably can be explained that GSK-3β increased PP2A activity via methylation of PP2A -in turn, dephosphorylated GSK-3β. Furthermore, Ser 404 and Ser 396, among tau phosphorylation sites, were the least favorable sites for PP2A in vitro [29,30]. Tau proteins have been believed as promising candidate biomarkers for Alzheimer-type neurodegenerations.
There are six different tau isoforms produced from a single gene in the adult human brain with high heterogeneity [11]. The increases in total tau and/or phosphorylated tau protein levels in cerebrospinal fluid are thought to be associated with neuronal cell death, releasing tau-related proteins into the extracellular cerebrospinal fluid [31]. We noticed that ketamine with or without ethanol treatment promoted elevated total tau protein expressions in the hippocampus and striatum, which tended to neurotoxicity.

Effect of Different Ketamine Treatments on Aβ Deposits
Aβ induces tau pathology in AD, [32] the pathological accumulation of Aβ indicates an imbalance between Aβ biosynthesis and clearance. Ketamine may reduce Aβ degradation by suppressing neprilysin expression in primary cultured astrocytes [13]. The current study supports the mechanism by which we observed extreme elevation of Aβ deposition in the hippocampal CA3 field and striatum of rats with different ketamine treatments.
Significantly, co-administration of ketamine with ethanol for 3 months promoted the most considerable increase of Aβ expression in the striatum (increased by 91.4%), accompanied by the elevation of BACE1 mRNA transcription. BACE1 is the crucial enzyme that initiates Aβ accumulation, and the activity of BACE1 is the ratelimiting step in APP processing to generate Aβ. 28 However, the inhibitions of BACE1 in the hippocampus and striatum of rats with different ketamine treatments for 6 months were insufficient to explain the increased Aβ levels fully. In this study, 6 months of drug administration probably promoted the other membrane protein activity involved in Aβ production, such as presenilins 1 and 2, [33], which remains mostly elusive. The over-inhibition of NMDAR activity may also result in neuronal degeneration or death [13]. The level of NMDAR1 subunit mRNA decreased in selective regions of the hippocampus and adjacent cortical areas of AD brains. 9 Reductions in NMDAR1 expressions in the CA3 from the hippocampus of different ketamine treatment groups (3 months) confirmed this hypothesis. Contrary to the results found in AD cases, different ketamine treatment for longterm promoted appreciable increases of NMDAR1 expression in the striatum of rats. We hypothesized that repeated ketamine treatments or ketamine and ethanol co-treatment induced compensatory upregulation of NMDAR expression that allowed the accumulation of toxic levels of intracellular Ca 2+ , leading to neurodegeneration [8]. It also could give a better understanding of the increase in NMDAR1 expression in the CA3 from the hippocampus of the K+E group (6 months). Ketamine exposed for 3 months stimulated NMDA2B overexpression in the hippocampus, complying with the published results that promoting adult hippocampal neurogenesis by ketamine is ventral dominant via elevation of GluN2B expression [34].

Conclusion
The present study demonstrates that region-specific AD-related biological events may damage the brain induced by recreational ketamine abuse. There are still limitations of the proposed possible mechanisms of tau hyperphosphorylation and extracellular Aβ deposits that need to be explored in detail.
Our study might open a new scenario for the molecular mechanism of cognitive dysfunction induced by recreational ketamine abuse by drawing on the AD investigation's achievements.